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In Vitro Genetic Toxicity

In vitro gene mutation in bacteria (Ames test)

Weight of evidence approach was used to fulfil the guideline requirement for this endpoint. In vitro bacterial mutagenicity study conducted for the substance, in vitro gene mutation study and in vivo cytogenicity study conducted for the read-across substance were used to evaluate genetic toxicity of the renewable hydrocarbons of wood origin (diesel type fraction).

Ames study by Thompson, P. W. (2012) is considered reliable without restrictions as the study was performed in compliance with GLP and according to OECD guideline 471. This study was conducted by using four strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537) and one strain of E. coli (WP2) with and without metabolic activation. Renewable hydrocarbons of wood origin (diesel type fraction) was tested for its ability to induce mutations at the concentration of 5, 15, 50, 150, 500, 1500, 5000 micrograms/plate. Test item did not produce any significant increase in mutation frequency in any of these strains, with any dose of the test item, either in the absence or presence of metabolic activation. Based on the study results the renewable hydrocarbons of wood origin (diesel type fraction) is considered to be non-mutagenic.

In vitro gene mutation in mammalian cells

This UVCB substance is a complex mixture of hydrocarbons. When there is no data on the substance itself the read-across data was also used to avoid unnecessary animal testing. The read-across from fossil diesel fuels is justified because the toxicokinetics and toxicological properties of renewable diesel are considered similar to fossil diesel fuels based on the similar composition and physical-chemical properties. The read-across justification and the data matrices are presented in annex 1 of the CSR.

The potential of the renewable hydrocarbons of wood origin (diesel type fraction) to induce gene mutations was assessed based on the in vitro gene mutation study (similar to OECD 476) conducted for the read-across substance, hydrodesulfurised middle distillate (API, 1986). In the study (API, 1986), mouse lymphoma cell line, L5178Y, was exposed to the test material, hydrodesulfurised middle distillate in ethanol, in the presence and absence of rat liver S-9 for four hours. In the inactive system (no S-9), the cells were exposed to test material at concentrations ranging from 25 nl/ml to 200 nl/ml and in the active system (with S-9) the cells were exposed to test material at concentrations ranging from 12.5nl/ml to 300 nl/ml. In addition to treated cells, a solvent control group and positive controls consisting of ethylmethane sulfonate for inactive studies (no S-9) and 3-methylcholanthrene (MCA) for active studies (with S-9) were also used to test the efficacy of the mutagenicity assay. This assay was performed in two sets to ensure reproduction of experimental results.

In cells treated with the test material in the absence of S-9, a wide range of toxicities was noted, including high toxicity at higher concentrations (value not specified). Additionally, a repeatable increase in mutant frequency was noted, but this frequency was not dose-dependent. In cells treated with the test material in the presence of S-9 activation, toxicity was again noted at various concentrations (values not specified) along with repeatable increases in mutant frequency. These mutant frequencies, however, were not dose-dependent. Based on these results, the study authors concluded that hydrodesulfurised middle distillate is weakly mutagenic in the mouse lymphoma assay, both in the presence and absence of S-9 activation.

In Vivo Genetic Toxicity

Read-across rat bone marrow micronucleus assay (API, 1985) is used to evaluate the in vivo cytogenicity of the renewable hydrocarbons of wood origin (diesel type fraction). Male and female rats were treated with a single intraperitoneal dose of a straight-run middle distillate at dose levels of 0, 300, 1000 and 3000 mg/kg bw. Bone marrow cells were harvested at 6, 24, and 48 hours post-treatment. There were no clinical signs of toxicity during the study, although male rats receiving 3000 mg/kg experienced 4% weight-loss at 24 hours after test substance administration, and female rats receiving 3000 mg/kg experienced 2% weight-loss at 48 hours. The positive control, triethylenemelamine (TEM), induced the appropriate response. There was no evidence of increased incidence of chromatid or chromosome gaps and breaks, fragments, structural rearrangements, or ploidy in bone marrow cells treated with straight-run middle distillate in comparison to controls.

Summary

Read-across substance, hydrodesulfurised middle distillate, showed weak mutagenicity potential in in vitro test. This finding has been regarded questionable since this observation is inconsistent with the several other studies using the same substance. Furthermore, no dose dependency was observed in the study either with or without metabolic activation.

The mechanism of PAH induced carcinogenesis is believed to be via the binding of PAH metabolites to DNA. A mutation may occur when these PAH metabolite-DNA adducts interfere with the regulation of cell differentiation and growth. The PAH analysis was conducted for the target UVCB substance to investigate whether it contains PAH compounds. The following PAHs were analysed naphthalene, 1-nethylnaphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene and indeno(1,2,3,c,d)pyrene. The results show that no other PAHs than naphthalene and pyrene were detected in the substance. The concentration of naphthalene and pyrene was 0.0015% (w/w) and 0.00034% (w/w), respectively. The detection limit of the analysis for individual PAH compound was 0.00024 % w/w.

In conclusion there is not enough evidence to classify this substance as genotoxic at this stage of CSA.


Justification for selection of genetic toxicity endpoint
No single study was selected since the hazard conclusion is based on the following three studies; Ames test (OECD 471), read-across in vitro gene mutation study (OECD 476) and read-across in vivo cytogenicity study (OECD 475).

Short description of key information:
Ames test (OECD 471): non-mutagenic with or without metabolic activation
In vitro gene mutation assay (OECD 476): weakly mutagenic with or without metabolic activation
In vivo cytogenicity assay (OECD 475): negative with or without metabolic activation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the Ames test conducted for this substance, and in vitro mutagenicity study and in vivo cytogenicity study conducted for the read-across substance, the renewable hydrocarbons of wood origin (diesel type fraction) is not classified as mutagenic in accordance with the criteria of CLP Regulation 1272/2008 and the EU directive 67/548/EEC.